Expanded Bed Affinity Selection

ABSTRACT

Separation of materials is achieved using affinity binding and acoustophoretic techniques. A column provided with a fluid mixture of materials for separation and support structures may be used with acoustic waves to block flow of the support structures. The support structures can have an affinity for one or more materials in the fluid mixture. By blocking flow of the support structures, materials bound or adhered to the support structure are also blocked.

CLAIM OF PRIORITY

This application claims priority under 35 USC § 119(e) to U.S. PatentApplication Ser. No. 62/490,574, filed on Apr. 26, 2017. Thisapplication is a continuation-in-part application of and claims priorityto U.S. patent application Ser. No. 15/222,800, filed on Jul. 28, 2016,which claims priority to 62/197,801, filed on Jul. 28, 2015. The entirecontents of each application is hereby incorporated by reference.

BACKGROUND

Separation of biomaterial has been applied in a variety of contexts. Forexample, separation techniques for separating proteins from otherbiomaterials are used in a number of analytical processes.

Acoustophoresis is a technique for separating particles and/or secondaryfluids from a primary or host fluid using acoustics, such as acousticstanding waves. Acoustic standing waves can exert forces on particles ina fluid when there is a differential in density and/or compressibility,known as the acoustic contrast factor. The pressure profile in astanding wave contains areas of local minimum pressure amplitudes atstanding wave nodes and local maxima at standing wave anti-nodes.Depending on their density and compressibility, the particles can bedriven to and trapped at the nodes or anti-nodes of the standing wave.Generally, the higher the frequency of the standing wave, the smallerthe particles that can be trapped.

SUMMARY

This disclosure describes technologies relating to methods, systems, andapparatus for acoustic separation of materials. The materials beingseparated may be biomaterials. The separation may employ materialsupport structures. The support structures may be beads. Afunctionalized material may be applied to the support structures thathas an affinity for one or more materials to be separated. The supportstructures may be mixed in a fluid that contains the materials. Thefluid mixture may be provided to a fluid column or flow chamber. Thesupport structures can be retained in the column against a fluid orfluid mixture flow through the column by provision of an acousticstanding wave at one end of the column that can prevent the supportstructures from passing.

In some examples, methods, systems, and apparatuses are disclosed forseparation of biomaterials accomplished by functionalized materialdistributed in a fluid chamber that bind the specific target materials.The specific target materials can be particles, including cells,recombinant proteins and/or monoclonal antibodies. The functionalizedmaterial, which may be beads and/or microcarrier structures are coatedor otherwise provided with an affinity material for attracting andbinding the specific target materials. The affinity material may be aprotein, ligand or other material that can form a bond with the targetmaterial.

In some example implementations, the affinity material and the targetmaterial can form antigen-antibody interactions with binding sites onthe functionalized material. In some instances, the target materialbecome bound to the functionalized material when a ligand of the targetmaterial or the functionalized material is conjugated to a matrix on thecomplementary material. The functionalized material includesfunctionalized microbeads. The functionalized microbeads include aparticular antigen ligand that has affinity for a correspondingantibody.

In some examples, material adhered to the support structures with thefunctionalized material remains in the column, while other free materialin the fluid may pass through the acoustic standing wave to provideseparation of materials. The support structures may be implemented tohave a certain acoustic contrast factor based on their density,compressibility, size or other characteristics that permits the supportstructures to react more strongly to the acoustic standing wave thanother materials in the fluid mixture.

The support structures may be agitated in the column to enhance theaffinity process. In different modes, the column fluid mixture thatpasses through the acoustic standing wave can be recirculated to thecolumn or not. The fluid flow in the column can be controlled to flow ornot, and when flowing, the rate of flow can be controlled. The fluid maybe stationary in the column and may have other processes appliedthereto, such as temperature adjustment, agitation, incubation, and/orany other useful process. The volume of the column can be effectivelymodified, such as with the provision of a plunger or piston in thecolumn. Heating or cooling can be applied to the column or the contentsof the column, internally or externally to the column.

The particulates may include beads, and wherein at least one of thebeads comprises a sphere with a diameter of about 20 to 300 μm andcomprises at least one of DEAE (N, N-diethylaminoethyl)-dextran, glass,polystyrene plastic, acrylamide, collagen, or alginate. Thecell-supporting material may include microbubbles that have a surfacecoating for growth of the cells. The cells may include, for example,T-cells, MRC-5 cells or stem cells.

An acoustic transducer can be used to generate the acoustic standingwave, which can generate pressure forces in one or multiple dimensions.In multiple dimensions, the acoustic standing wave forces can be of thesame order of magnitude. For example, forces in the direction of wavepropagation may be of the same order of magnitude as forces that aregenerated in a different direction. An interface region can be generatednear a border of the acoustic standing wave that contributes topreventing support structures from passing. Multiple transducers may beused, some for generating an acoustic wave in one or modes, and othersfor generating an acoustic wave in another, different mode. For example,the acoustic wave can be a standing wave that can generate pressureforces in one dimension or in multiple dimensions. The acoustic wave canbe generated in a mode to form an interface region to prevent passage ofcertain materials while permitting passage of other materials. Theacoustic wave can be generated in a mode to trap and cluster certainmaterials that build in size until the gravity or buoyancy forces on theclusters surpass the other forces on the clusters, such as fluidic oracoustic forces, so that the clusters drop or rise out of the acousticwave.

Collecting cells may be performed with or without turning off theacoustic transducer. An additive which enhances aggregation of thesupport structures into the flow chamber may be applied. The method mayfurther include recirculating the support structures, such as beads, toa culturing chamber coupled to the flow chamber. The method may alsoinclude processing the collected cells for infusion into a subjectpatient. Subsequent to preferentially trapping, the method may includeallowing the trapped cells and/or cell-supporting material to rise orsettle out of the fluid due to a buoyance or gravity force. The risingor settling cells and/or support material may exit the flow chamber. Themode of trapping cells or support material for separation by rising orsettling out of the fluid may be accompanied by a mode of preventing orpermitting the cells and/or support material from passing through afluid path. The mode of preventing or permitting passage may beimplemented with an acoustic wave with an interface region across thefluid path.

In some example implementations, the material includes target compounds,such as recombinant proteins and monoclonal antibodies, viruses, and/orlive cells (e.g., T cells). Beads or microcarriers with or withoutfunctionalized material on their surfaces may be the target compounds orcomponents.

An example apparatus may include a flow chamber configured to receivefluid containing functionalized material. The flow chamber may be in theform of a column. An acoustic transducer is arranged in relation to theflow chamber, for example, acoustically coupled to the flow chamber, toprovide an acoustic wave or signal into the flow chamber when excited.Excitation of the transducer can generate a multi-dimensional acousticfield inside the chamber that includes first spatial locales whereacoustic pressure amplitude is elevated from a base level, such as, forexample when the acoustic transducer is turned off, and second spatiallocales where acoustic pressure amplitude has little or no elevationfrom the base level, for example the acoustic pressure amplitude may beequivalent to that when the acoustic transducer is turned off.

In some modes, the functional material may be driven to and retained atthe first or second locales of the multidimensional acoustic field. Inother modes, the functional material may be prevented from entering themultidimensional acoustic field in accordance with an edge effect at aninterface region. Materials to be processed that include targetmaterials for separation may be flowed into the flow chamber wherefunctionalized material is retained such that a portion of the targetmaterials with features complementary to the functionalized materialbecome bound to the functionalized material while other portions of thematerials pass through the flow chamber. The chamber may be configuredfor vertical flow which may be in an upward or downward direction. Fluidpaths to the chamber may be provided at a top and/or bottom of thechamber. An acoustic transducer can be coupled to a top and/or bottom ofthe chamber to generate an acoustic field at that locale.

The functionalized microcarriers may also be circulated after therecombinant proteins or monoclonal antibody is eluted from the surfaceby a buffer or other process elution. This allows for greater surfacearea and affinity interaction of the functionalized microcarriers withthe expressed proteins from the bioreactor, increasing the efficiency ofthe acoustic fluidized bed chromatography process.

In some example implementations, the apparatus provides functionalizedparticles, such as beads, in an arrangement that provides more spacebetween particles, such as beads or cells, than packed columns. Thelower density decreases the likelihood that non-target biomaterials willclog flow paths between the functionalized particles. In some exampleimplementations, recirculating media containing the target biomaterialsin effect increases the capture surface area of the apparatus by passingfree target biomaterials past the functionalized particles multipletimes. The reduced contact of non-target biomaterials such as cells canhelp preserve the viability of cells. The technology described here canbe used in high- or low-density cell culture, new research applications,large production culture volumes, e.g., more than 1,000 liters,efficient monitoring and culture control, reduction of costs andcontamination in cell culture applications.

The details of one or more implementations of the subject matterdescribed in this specification are set forth in the accompanyingdrawings and the description below. Other features, aspects, andadvantages of the subject matter will become apparent from thedescription, the drawings, and the claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The disclosure is described in greater detail below, with reference tothe accompanying drawings, in which:

FIG. 1 is a simplified diagram of an acoustic affinity process;

FIG. 2 is a side elevation view of an acoustic affinity system operatedin an edge effect mode;

FIG. 3 is a side elevation view of an acoustic affinity system operatedin a cluster mode;

FIG. 4 is a photograph of a front elevation view of a fluidized bed setup;

FIG. 5 is a diagram of an acoustic affinity system and process;

FIG. 6 is a diagram of an acoustic affinity system and process;

FIG. 7 is a diagram of an acoustic affinity system and process;

FIG. 8 is a diagram of an acoustic affinity system and process;

FIG. 9 is a diagram of an affinity positive selection in an acousticaffinity process;

FIG. 10 is a diagram of an affinity negative selection in an acousticaffinity process;

FIG. 11 is a graph showing the retention versus inflow fluid rate;

FIG. 12 is a graph showing the cell viability versus column volumes;

FIG. 13 is a graph showing a histogram of particle sizes;

FIG. 14 is a diagram of an acoustic affinity system and process with therecirculation; and

FIG. 15 is a bar graph showing purity and recovery in a recirculationarrangement.

DETAILED DESCRIPTION

This disclosure describes methods, systems and apparatuses that employan acoustic standing wave with nodes and antinodes to separate supportstructures such as beads or coated microbubbles from other materials ina chamber such as a column. The example implementations described hereinmay be operated in different modes. For example, in some modes, anacoustic wave is generated with certain characteristics across thechamber. The acoustic wave may be generated by an acoustic transducer,which may be located at one end of the column. The acoustic wave maycause an interface region to be generated that blocks certain materialsfrom entering the acoustic wave, while permitting passage of othermaterials. The acoustic wave characteristics can be controlled to blockor pass materials based on parameters such as compressibility, density,size, acoustic contrast factor, and any other parameter that isresponsive to the acoustic waves. In other modes, an acoustic wave isgenerated with spatial locales that capture materials to form clustersthat increase in size to a point where the gravity or buoyancy force onthe cluster exceeds that of the acoustic or fluid drag force, causingthe cluster to exit the acoustic wave.

The modes discussed herein may be employed together or separately or incombination. The modes may be employed or generated with one or moreacoustic transducers. The acoustic field generated by the acoustic wavecan be configured to block or permit passage of certain materials. Forexample, support structures for cells, which may be in the form ofbeads, bead/cell complexes or particles, may be blocked from passagethrough the acoustic field. Materials such as cells may be passedthrough the fluid chamber. The support structures include functionalizedmaterial that can bind with at least some of the material passed throughthe fluid chamber. The material that is bound to the support structuresvia the functionalized material is retained in the fluid chamber by thesupport structures being retained in the fluid chamber with the acousticwave. Material that is not bound to the support structures may pass outof the fluid chamber through the acoustic wave. The technique of usingacoustic waves to perform affinity separation obtains a number ofadvantages as described in more detail herein.

Referring to FIG. 1, a diagram illustrates an acoustic affinity process100. Functionalized beads 102 are placed in a chamber 104 that containstargeted and non-targeted material. The target material corresponds tothe functionalization provided to beads 102. Process 100 illustrates thetarget material being bound to beads 102 in an affinity binding process.Beads 102 are collected or influenced by an acoustic standing wavegenerated by transducer 106 between transducer 106 and reflector 108.The remaining material in chamber 104 can be removed by flowing fluidthrough chamber 104 while beads 102 are retained by the acoustic wave.Process 100 illustrated in FIG. 1 can be a positive or negativeselection process, where the target material is desired to be itselfcollected or removed from the other materials, respectively.

In accordance with some examples, an acoustic affinity system isimplemented that can include in the features of being closed, automatedand/or single-use. The system can be considered closed if the componentscan be sealed from an open-air environment. An automated system is ableto operate autonomously, with little or no operator intervention. Thesystem is single use when components and materials employed for anaffinity separation run, which may include multiple recirculations, aredisconnected and discarded after the an affinity separation run. Asingle use system can avoid the additional steps of cleaning andsterilizing the equipment components and materials for subsequent runs.

Previous systems for affinity separation employed magneticallyresponsive beads. These beads may incur challenges during manufacturingprocesses as they do not dissolve or are not readily consumed in vivoand are preferentially completely removed from any treatment supplied toa patient. While such beads may be used in the present acoustic affinityseparation system, the use of acoustics offers the possibility for theuse of support structures, such as beads, that are tailored to bespecifically acoustically responsive. For example, the beads can benonmagnetic or non-magnetically responsive, and highly acousticallyresponsive. The acoustically responsive beads can be composed of avariety of materials, significantly increasing the flexibility of theprocessing system in which they are employed. These acoustic affinitybeads can be composed of dissolvable material that is biocompatible,which can alleviate aggressive bead removal processes that are employedwith magnetically responsive beads.

The acoustic affinity system can be configured to have increasedthroughput compared with current systems. For example, the fluid flowrate through the system can be increased over that typically used withconventional affinity systems. The system can be configured with largerchannels that permit higher flow rates and volumes. The expansion of thecell population can be implemented within the presently disclosedsystems or can be implemented externally and fed to the acousticaffinity system.

The configuration of the acoustic affinity system permits the use ofmultiple types of support structures or beads that may have differentcharacteristics, such as different ranges of sizes or densities. Thedifferent groups of support structures or beads may be provided withdifferent types of functionalized material such as proteins, antigens orantibodies to thereby enable multiplexing of affinity separation. Thisconfiguration permits complex, single-pass affinity selections to berealized.

In some example implementations, a column is provided with a volume ofbeads that have an affinity for a certain type of cell. Cells introducedinto the column form a complex with the beads, which complexes can beseparated from the column volume using acoustic techniques. Theseparation may be leveraged to harvest cultured cells of interest, andthe extracted cells may be infused into a patient. Using acoustics withan affinity binding system to separate cultured cells of interest can beapplicable to a variety of cell therapy applications, e.g., vaccinetherapies, stem cell therapies, particularly allogenic and autologoustherapies, or regenerative therapies.

An acoustic wave is generated in a flow chamber, such as a column, toeffectuate separation of beads and bead complexes from unbound cells ormaterials in a fluid. The separation can be negative or positive, wherethe unwanted material to be excluded is bound to the beads, or where thematerial desired in the separation is bound to the beads, respectively.The material of interest, for either negative or positive selection, maybe different types of cells, including adherent cells. Example adherentcells may include human multipotent stem cells (hMSC), human mesenchymalstem cells (also hMSC), human pluripotent stem cells (hPSC), humandermal fibroblasts (hDF), human chondrocytes, and some T lymphocytes.Adherent cells may differ in their antigen specificity (e.g. CD8adherent cell). The lines used in cell therapy may be mono- orpolyclonal (e.g. polyclonal CD8 adherent cell line), and CAR (chimericantigen receptor) adherent cells (a.k.a. artificial adherent cellreceptors, or chimeric adherent cell receptors, or chimericimmunoreceptors. These are T-cells modified to recognize a specificprotein. The beads employed in the acoustic affinity separation systemcan be configured to bind or not bind to these cells or material ofinterest for negative or positive selection.

The bead technology described here can be used in high density cellculture, new research applications, large production culture volumes,e.g., more than 1,000 liters, efficient monitoring and culture control,reduction of costs and contamination in cell culture applications. Thebeads used may be commercially available, such as the MAGNE magneticaffinity beads or polystyrene beads supplied by Promega Corporation orMACS beads supplied by Miltenyi Biotec. The size of the beads, forexample their diameter, may be in the nanometer or micrometer range.Cospheric beads may be used, which are beads with at least two layers.The layers may have different characteristics, such as differingcontrast factors, structural rigidity, or any other characteristics thatare desired to be combined in a single bead through the use of multiplelayers.

Some implementations may use microbubbles as support structures to bindmaterial of interest. The microbubbles can be composed by a shell ofbiocompatible materials and ligands capable of linking to the cells ormaterial of interest, including proteins, lipids, or biopolymers, and bya filling gas. Low density fluids may be used for relative ease ofmanufacturing. The microbubble shell may be stiff (e.g., denaturatedalbumin) or flexible (phospholipids) and presents a thickness from 10 to200 nm. The filling gas can be a high molecular weight andlow-solubility filling gas or liquid (perfluorocarbon or sulfurhexafluoride), which can produce an elevated vapor concentration insidethe microbubble relative to the surrounding fluid, such as blood, andincrease the microbubble stability in the peripheral circulation. Themicrobubble shell can have a surface coating such as a lipid layer. Thelipid layer may be utilized as scaffolds or substrates for materialgrowth such as cells or biomolecules. Active groups may be easier toconjugate directly to the glass surface. The microbubbles may have adiameter in a range of 2 to 6 micrometers. The coated microbubbles mayhave a negative contrast factor.

Examples discussed above provide beads as support structures. Othersupport structures such as coated bubbles or microbubbles can be alsoused. For the sake of convenience, support structures may be referred toherein collectively as beads, which term is intended to encompass alltypes of support structures, including beads, bubbles, microcarriers andany other type of affinity material that can bind to or be bound to atarget material of interest.

Cells are bound to beads, e.g., CD3/CD28 activated beads. As discussedin further detail below, the beads can be functionalized with surfacechemistry such that the cells or material of interest can be attached toor adherent to the surface of the beads. The beads can include supportmatrices allowing for the growth of adherent cells in bioreactors orother cell culturing systems. In some cases, adherent cells will bind tothe beads without the antigens on the surface and the beads can befunctionalized or non-functionalized.

Structurally, the beads include spheres with a diameter in a range of 1to 300 μm, e.g., in the range of 125 to 250 The spheres can havedensities in a range of 1.02-1.10 g/cm³. In some instances, the beadscan also include rod-like structures. The beads may be smooth ormacroporous.

The core of the beads can be made from different materials, such asglass, polystyrene plastic, acrylamide, collagen, and alginate. The beadmaterials, along with different surface chemistries, can influencecellular behavior, including morphology and proliferation.

The beads can be coated with a variety of coatings such as glass,collagen (e.g., neutral or charged gelatin), recombinant proteins orchemical treatments to enhance cell attachment, which may lead to moredesirable cell yields for a number of different cell lines.

Surface chemistries for the beads can include extracellular matrixproteins, recombinant proteins, peptides, and positively or negativelycharged molecules. The surface charges of the micro carriers may beintroduced from a number of different groups, including DEAE (N,N-diethylaminoethyl)-dextran, laminin or vitronectin coating (extracellular matrix proteins). In the DEAE-dextran example, a mild positivecharge can be added to the surface.

In some implementations, the beads are formed by substituting across-linked dextran matrix with positively charged DEAE groupsdistributed throughout the matrix. This type of bead can be used forestablished cell lines and for production of viruses or cell productsfrom cultures of primary cells and normal diploid cell strains.

In some implementations, the beads are formed by chemically coupling athin layer of denatured collagen to the cross-linked dextran matrix.Since the collagen surface layer can be digested by a variety ofproteolytic enzymes, it provides opportunities for harvesting cells fromthe beads while maintaining increased or maximum cell viability andmembrane integrity. The acoustic affinity system discussed herein can beoperated with a number of types of beads, three general groupings ofwhich are discussed below.

The beads may be constructed and configured according to cGMP (currentgood manufacturing practice) standards or regulations. One example groupof beads that may be used in the acoustic affinity system are large,dense beads. These large beads may possess the followingcharacteristics.

-   -   Non-magnetic    -   Average size of about 50 um    -   Slower binding kinetics    -   More easily separated using acoustic techniques    -   Positive acoustic contrast factor    -   Dissolvable and biocompatible    -   poly(lactic-co-glycolic acid), PLGA    -   Not internalized by cells

Another example group of beads are those referred to herein as mediumsized beads. These medium sized beads may possess the followingcharacteristics.

-   -   Non-magnetic    -   Average size in the range of about 1-10 um    -   Dissolvable and biocompatible    -   Binding kinetics faster than large beads    -   Use large acoustic contrast    -   Negative and positive contrast    -   PLGA or proprietary lipid-based

Another example group of beads are those referred to herein as smallbeads. These small beads may possess the following characteristics.

-   -   Non-magnetic    -   Average size in the range of about 200 nm-2 um    -   Dissolvable and biocompatible    -   Very fast binding    -   Separation through clustering    -   Negative contrast factor, low speed of sound & high density    -   Proprietary lipid-based

Different types of beads may be chosen for different types ofapplications. For example, larger beads may be used when the cells arecultured with the beads, or when the affinity binding takes place in anon-flowing mode.

The beads used for the affinity binding can be held back by or passedthrough an acoustic wave generated by an acoustic transducer. Theacoustic transducer may generate a multi-dimensional acoustic standingwave in a flow chamber to create an acoustic field that includes localesof increased pressure radiation forces. The acoustic transducer caninclude a piezoelectric material that is excited to vibrate and generatean acoustic wave. The acoustic transducer can be configured to generatehigher order vibration modes. For example, the vibrating material in theacoustic transducer can be excited to obtain a standing wave on thesurface of the vibrating material. The frequency of vibration isdirectly related to the frequency of the excitation signal. In someimplementations, the vibrating material is configured to have an outersurface directly exposed to a fluid layer, e.g., the fluid or mixture ofbeads and cultured cells in a fluid flowing through the flow chamber. Insome implementations, the acoustic transducer includes a wear surfacematerial covering an outer surface of the vibrating material, the wearsurface material having a thickness of a half wavelength or less and/orbeing a urethane, epoxy, or silicone coating, polymer, or similar thincoating. In some implementations, the acoustic transducer includes ahousing having a top end, a bottom end, and an interior volume. Thevibrating material can be positioned at the bottom end of the housingand within the interior volume and has an interior surface facing to thetop end of the housing. In some examples, the interior surface of theacoustic material is directly exposed to the top end housing. In someexamples, the acoustic transducer includes a backing layer contactingthe interior surface of the acoustic material, the backing layer beingmade of a substantially acoustically transparent material. One or moreof the configurations can be combined in the acoustic transducer to beused for generation of a multi-dimensional acoustic standing wave.

The generated multi-dimensional acoustic standing wave can becharacterized by strong gradients in the acoustic field in alldirections, not only in the axial direction of the standing waves butalso in lateral directions. In some instances, the strengths of suchgradients are such that the acoustic radiation force is sufficient toovercome drag forces at linear velocities on the order of mm/s.Particularly, an acoustic radiation force can have an axial forcecomponent and a lateral force component that are of the same order ofmagnitude. As a consequence, the acoustic gradients result in strongtrapping forces in the lateral direction.

The multi-dimensional acoustic standing wave can give rise to a spatialpattern of acoustic radiation force. The multidimensional acousticstanding wave may be generated from one transducer and reflector pairdue to the multimode perturbations of the piezoelectric material in thetransducer. The acoustic radiation force can have an axial forcecomponent and a lateral force component that are of the same order ofmagnitude. The spatial pattern may manifest as periodic variations ofradiation force. More specifically, pressure node planes and pressureanti-node planes can be created in a fluid medium that respectivelycorrespond to floor acoustic radiation force planes with maximum andminimum acoustic radiation force planes in between pressure nodal andanti-nodal planes. Pressure nodal planes are also acoustic displacementanti-nodal planes, and vice versa. The spatial pattern may function muchlike a comb filter in the fluid medium.

In some modes, discussed in greater detail below, the spatial patternmay create an interface region that blocks entry of particles withcertain characteristics from entering or crossing the acoustic wave. Inother modes of operation, discussed in greater detail below, the spatialpattern may be used to trap particles, for example, of a particular sizeor size range, while particles of a different size or size range may notbe trapped. The modes may be employed separately or together incombination to provide both a barrier and trapping function, in the sameor separate locale.

In a multidimensional acoustic standing wave, the acoustic radiationforces within a particular pressure nodal plane are such that particlesare trapped at several distinct points within these planes. The trappingof particles leads to the formation of cluster of particles, whichcontinuously grow in size, and, upon reaching a critical size, settleout or rise out of the primary fluid continuously because of enhancedgravitation or buoyancy settling. For example, the spatial pattern canbe configured, for example, by adjusting the insonification frequencyand/or phase, power, voltage and/or current supplied to the transducer,or fluid velocity or flow rate, to allow the cultured cells to freelyflow through while trapping the support structures, such as beads ormicrobubbles, thereby separating at least the trapped support structuresfrom cells or other materials in the fluid.

In some example implementations, one or more multi-dimensional acousticstanding waves are generated between an ultrasonic transducer and areflector. An acoustic wave is continually launched from the acoustictransducer and reflected by the reflector to interfere with the launchedacoustic wave to form an acoustic standing wave. The formation of theacoustic standing wave may depend on a number of factors, includingfrequency, power, medium, distance between the transducer and reflector,to name a few. The standing wave can be offset at the transducer or thereflector so that local minima or maxima are spaced from the transduceror from the reflector. The reflected wave (or wave generated by anopposing transducer) can be in or out of phase with the transducergenerated wave. The characteristics of the standing wave can be modifiedand/or controlled by the drive signal applied to the transducer, such asby modifying and/or controlling the phase, amplitude or frequency of thedrive signal. Acoustically transparent or responsive materials may alsobe used with the transducer or reflector to modify and/or control thestanding wave.

As the fluid mixture flows between an ultrasonic transducer andreflector, or two facing ultrasonic transducers, between which one ormore multi-dimensional acoustic standing waves are established,particles or secondary fluid cluster, collect, agglomerate, aggregate,clump, or coalesce. The clustering of material may take place at thenodes or anti-nodes of the multi-dimensional acoustic standing wave,depending on the particles' or secondary fluid's acoustic contrastfactor relative to the host fluid. The particles form clusters thateventually exit the multi-dimensional acoustic standing wave nodes oranti-nodes when the clusters have grown to a size large enough toovercome the holding force of the multi-dimensional acoustic standingwave. For example, the clusters grow in size to a point where thegravity or buoyancy forces become dominant over the acoustic or fluiddrag forces, causing the clusters to respectively sink or rise. Forfluids/particles that are denser than the host fluid, such as is thecase with most cells, the clusters sink and can be collected separatelyfrom the clarified host fluid. For fluids/particles that are less densethan the host fluid, the buoyant clusters float upwards and can becollected.

The scattering of the acoustic field off the particles creates secondaryacoustic forces that contribute to driving particles or fluid dropletstogether. The multi-dimensional acoustic standing wave generates athree-dimensional acoustic radiation force, which acts as athree-dimensional trapping field. The acoustic radiation force isproportional to the particle volume (e.g. the cube of the radius) whenthe particle is small relative to the wavelength. The force isproportional to frequency and the acoustic contrast factor. The forcescales with acoustic energy (e.g. the square of the acoustic pressureamplitude). When the acoustic radiation force exerted on the particlesis stronger than the combined effect of fluid drag force and buoyancyand gravitational force, the particles are trapped within the acousticstanding wave. The particle trapping in a multi-dimensional acousticstanding wave results in clustering, concentration, agglomeration and/orcoalescence of the trapped particles. Relatively large solids of onematerial can thus be separated from smaller particles of a differentmaterial, the same material, and/or the host fluid through enhancedgravitational/buoyancy separation.

The multi-dimensional standing wave generates acoustic radiation forcesin a number of directions, including in the direction of acoustic wavepropagation and in a direction that is the lateral to the acoustic wavepropagation direction. As the mixture flows through the acousticchamber, particles in suspension experience a strong axial forcecomponent in the direction of the standing wave. Since this acousticforce is across (e.g. perpendicular to) the flow direction, it is notaligned with the fluid drag force. The acoustic force can thus quicklymove the particles to pressure nodal planes or anti-nodal planes,depending on the contrast factor of the particle. The lateral acousticradiation force acts to move the concentrated particles towards thecenter of each planar node, resulting in clustering, agglomeration orclumping. The lateral acoustic radiation force component can overcomefluid drag for such clumps of particles, to continually grow theclusters, which can exit the mixture due to dominant gravity or buoyancyforces. The drop in drag per particle as the particle cluster increasesin size, as well as the drop in acoustic radiation force per particle asthe particle cluster grows in size, may separately or collectivelyinfluence operation of the acoustic separator device. In the presentdisclosure, the lateral force component and the axial force component ofthe multi-dimensional acoustic standing wave are of the same ordifferent order of magnitude. In a multi-dimensional acoustic standingwave generated by a single transducer, the axial force can be comparablewith the lateral force. The lateral force of such a multi-dimensionalacoustic standing wave is much higher than the lateral force of a planarstanding wave, usually by two orders of magnitude or more.

The multi-dimensional acoustic standing wave generated for variousmodes, including to form a barrier or for clustering, is obtained byexciting a piezoelectric material at a frequency that excites afundamental 3D vibration mode of the transducer. The transducer may becomposed of various materials that may be perturbed to generate anultrasonic wave. For example, the transducer may be composed of apiezoelectric material, including a piezoelectric crystal orpoly-crystal. Perturbation of the piezoelectric material, which may be apiezoelectric crystal or poly-crystal, in the ultrasonic transducer toachieve a multimode response allows for generation of a multidimensionalacoustic standing wave. A piezoelectric material can be specificallydesigned to deform in a multimode response at designed frequencies,allowing for generation of a multi-dimensional acoustic standing wave.The multi-dimensional acoustic standing wave may be generated withdistinct modes of the piezoelectric material such as a 3×3 mode thatgenerates nine separate multidimensional acoustic standing waves. Amultitude of multidimensional acoustic standing waves may also begenerated by allowing the piezoelectric material to vibrate through manydifferent mode shapes. Thus, the material can be selectively excited tooperate in multiple modes such as a 0×0 mode (i.e. a piston mode), 1×1,2×2, 1×3, 3×1, 3×3, and other higher order modes. The material can beoperated to cycle through various modes, in a sequence or skipping pastone or more modes, and not necessarily in a same order with each cycle.This switching or dithering of the material between modes allows forvarious multidimensional wave shapes, along with a single piston modeshape to be generated over a designated time. The transducers may becomposed of a piezoelectric material, such as a piezoelectric crystal orpoly-crystal, which may be made of PZT-8 (lead zirconate titanate). Suchcrystals may have a major dimension on the order of 1 inch and larger.The resonance frequency of the piezoelectric material may nominally beabout 2 MHz and may be operated at one or more frequencies. Eachultrasonic transducer module may include single or multiple crystals.Multiple crystals can each act as a separate ultrasonic transducer andare can be controlled by one or multiple controllers, which controllersmay include signal amplifiers. The control of the transducer can beprovided by a computer control that can be programmed to provide controlsignals to a driver for the transducer. The control signals provided bythe computer control can control driver parameters such as frequency,power, voltage, current, phase, or any other type of parameter used toexcite the piezoelectric material. The piezoelectric material can besquare, rectangular, irregular polygon, or generally of any arbitraryshape. The transducer(s) is/are used to create a pressure field thatgenerates forces of the same order of magnitude in a lateral and anaxial direction.

In some examples, the size, shape, and thickness of the piezoelectricmaterial can determine the transducer displacement at differentfrequencies of excitation. Transducer displacement with differentfrequencies can be used to target certain material in an ensonifiedfluid. For example, higher frequencies with shorter wavelengths cantarget smaller sized material. Lower frequencies with longer wavelengthscan target smaller sized material. In these cases of higher and lowerfrequencies, material that is not influenced by the acoustic wave maypass through without significant change. Higher order modaldisplacements can generate three-dimensional acoustic standing waveswith strong gradients in the acoustic field in all directions, therebycreating strong acoustic radiation forces in all directions, whichforces may, for example be equal in magnitude, leading to multipletrapping lines, where the number of trapping lines correlate with theparticular mode shape of the transducer.

The piezoelectric crystals of the transducers described herein can beoperated at various modes of response by changing the drive parameters,including frequency, for exciting the crystal. Each operation point hasa theoretically infinite number of vibration modes superimposed, whereone or more modes are dominant. In practice, multiple vibration modesare present at arbitrary operating points of the transducer, with somemodes dominating at a given operating point.

Referring to FIG. 2, a system 200 operating in interface barrier mode isillustrated. An acoustic interface region 202 is employed to block beads204 from passing through acoustic wave 206. Acoustic wave 206 isgenerated by an acoustic transducer 208 continually launching anacoustic wave that is reflected by a reflector 210 to generate astanding wave with localized minima (nodes) and maxima (anti-nodes). Apressure rise may be generated on the upstream side of acoustic wave 206at interface region 202, along with an acoustic radiation force actingon the incoming suspended particles. Interface region 202, also referredto as providing an edge, boundary or barrier effect, can act as abarrier to certain materials or particles. In system 200, a majority, orsubstantially all, of beads 204 are prevented from entering acousticwave 206. Other materials can pass through interface region 202.Acoustic wave 206 is configured to influence beads 204, while othermaterial experiences a lower influence to permit them to pass throughacoustic wave 206.

Interface region 202 is located at an upstream bounding surface orregion of the volume of fluid that is ensonified by acoustic transducer208. For example, the fluid may flow across interface region 202 toenter the ensonified volume of fluid and continue in a downstreamdirection. The frequency of acoustic standing wave 206 may be controlledto have desired characteristics, such that, for example, differentcontrast factor materials may be held back by or allowed throughacoustic standing wave 206. Interface region 202 can be generated andcontrolled to influence, for example, particles of a first size range tobe retained. Acoustic standing wave 206 can be generated and controlledto permit, for example, particles of a second size range that isdifferent from the first to pass through. Acoustic standing wave 206that forms interface region 202 may be modulated so as to block or passselective materials. The modulation can be employed to block or passselective materials at different times while fluid flows through theacoustic field generated by acoustic standing wave 206.

In some example implementations, acoustic standing wave 206 produces athree-dimensional acoustic field, which, in the case of excitation bytransducer 208 implemented as a rectangular transducer, can be describedas occupying a roughly rectangular prism volume of fluid across thedirection of fluid flow. Acoustic wave 206 can be generated as astanding wave. The generation of acoustic wave 206 can be achieved withtwo transducers facing each other across the fluid flow. A singletransducer, e.g., transducer 208, may be used to launch acoustic wave206 through the fluid toward an interface boundary region that providesa change in acoustic properties, such as may be implemented with achamber wall or reflector 210. The acoustic wave reflected from theinterface boundary can contribute to forming a standing acoustic wavewith the acoustic wave launched from transducer 208. During operation atdifferent or changing flow rates, the location of interface region 202may move upstream or downstream.

The acoustic field generated by acoustic standing wave 206 exerts anacoustic radiation pressure (e.g., a pressure rise) and an acousticradiation force on the fluid and materials at interface region 202. Theradiation pressure influences material in the fluid to block upstreammaterials with certain characteristics from entering the acoustic field.Other materials with different characteristics than the blockedmaterials are permitted to pass through the acoustic field with thefluid flow. The characteristics that affect whether the materials orparticles are blocked or passed by the acoustic field include materialcompressibility, density, size and acoustic contrast factor. Theparameters that can influence the generation or modulation of theacoustic wave include frequency, power, current, voltage, phase or anyother drive parameters for operating transducer 208. Other parametersimpacting acoustic wave 206 include transducer size, shape, thickness,as well as chamber size and fluid parameters such as density, viscosityand flow rate.

Referring to FIG. 3, a system 300 operating in clustering mode isillustrated. One or more multi-dimensional acoustic standing waves 306are created between an ultrasonic transducer 308 and a reflector 310. Anacoustic wave is continually launched from acoustic transducer 308 andreflected by reflector 310 to interfere with the launched wave, therebyforming a standing wave 306 that has local minima and maxima, or nodesand anti-nodes, respectively. The reflected wave (or wave generated byan opposing transducer) can be in or out of phase with thetransducer-generated wave. The characteristics of the standing wave canbe modified and/or controlled by the drive signal applied to transducer308, such as by modifying and/or controlling the phase, amplitude orfrequency of the drive signal. Acoustically transparent or responsivematerials may also be used with transducer 308 or reflector 310 tomodify and/or control standing wave 306.

In a clustering mode, beads 304, bead complexes 314 and/or particlessuch as cells cluster, collect, agglomerate, aggregate, clump, orcoalesce within multi-dimensional standing wave 306. The clustering mayoccur at the nodes or anti-nodes of multi-dimensional acoustic standingwave 306, depending on the acoustic contrast factor of beads 304 or theparticles relative to the host fluid. For example, beads 304, beadcomplexes 314 or particles that have a positive acoustic contrast factorare driven to the nodes of multi-dimensional acoustic standing wave 306,while beads 304, bead complexes 314 or particles that have a negativeacoustic contrast factor are driven to the anti-nodes. The clusteredbeads 304, bead complexes 314 or particles form clusters 312 thateventually exit the nodes or anti-nodes of multi-dimensional acousticstanding wave 306 when clusters 312 have grown to a size large enough toovercome the holding force of multi-dimensional acoustic standing wave306. For example, as clusters 312 grow in size in multi-dimensionalacoustic standing wave 306, gravity or buoyancy forces begin to dominateover acoustic and/or fluid drag forces. Once the size of a cluster 312is large enough to cause the gravity or buoyancy forces on cluster 312to exceed the acoustic and/or fluid drag forces, cluster 312 exitsmulti-dimensional acoustic standing wave 306.

For beads 304, bead complexes 314 or particles that, for example, have apositive acoustic contrast factor, clusters 312 typically sink withgravity forces. For beads 304, bead complexes 314 or particles that, forexample, have a negative acoustic contrast factor, clusters 312typically rise with buoyancy forces. Gravity is not depicted in FIG. 3,and the orientation of system 300 can be with gravity aligned with oragainst the fluid flow direction. With gravity against the direction offluid flow, clusters 312 are depicted as sinking due to gravity forces.With gravity aligned with the direction of fluid flow, clusters 312 aredepicted as rising due to buoyancy forces.

In this mode of operation, beads 304, and bead complexes 314, areretained in the chamber by sinking or rising out of the acoustic wave.The beads tend to be lightly clustered in this mode and tend to beredistributed in the chamber to permit additional interaction withtarget material or cells. In addition, an agitator can be provided tothe chamber to promote movement and redistribution of the clusteredbeads.

Particles such as cell Type A are not captured in multi-dimensionalacoustic standing wave 306. The characteristics of the Type A cells andmulti-dimensional acoustic standing wave 306 permit the Type A cells topass without being captured and/or clustering. The Type B cells arebound to beads 304 to form bead complexes 314. Accordingly, Type B cellsmay themselves pass through multi-dimensional acoustic standing wave 306but may be driven into a cluster 312 if bound to beads 304.

Referring to FIG. 4, a set up for a fluidized bed system 400 isillustrated. The fluidized bed is composed of cospheric beads with arange of about 10% to about 30% packing. The acoustic transducer isattached to a top of the column housing the fluidized bed. Connectionsare provided at a base of the column for introducing or removing fluidthat may entrain beads, cells or other materials. The configuration andoperation of system 400 can be controlled with a controller that providesignals to operate a driver for the transducer, as well as fluid controldevices, such as pumps, valves or switches. The controller receivesfeedback from sensors, which can include turbidity sensors, fluid flowsensors and/or valve sensors. The controller also receives feedback fromthe acoustic transducer to contribute to providing a close looptransducer control. The different modes of operation of thetransducer(s) can be implemented by the controller. The controller canbe employed to provide automated operation for system 400 in accordancewith the examples discussed herein. For example, the controller can beprovided with a number of automation profiles from which an operator canselect to implement an automated acoustic affinity cell selectionprocess. As illustrated in FIG. 4, the acoustic transducer is employedin a mode to generate an edge effect or interface region as discussedabove. Testing on the throughput of the column with the transduceroperated in this mode has established some guidelines for flowvelocities or flow rates that can be employed in the column while thebeads are maintained in the column by the acoustic standing wave andedge effect.

Referring to FIG. 5, a fluidized bed system 500 is illustrated. In thisexample implementation, column 502 is packed with affinity beads 504,which may be in the range of about 10% to about 30% packing where %packing indicates the percentage of bead volume versus volume of theentire column. Beads 504 are provided with affinity structures to bindto target cells 506. An acoustic transducer 512 capable of generating anacoustic field is coupled to a top of column 502. In operation, a mix oftarget cells 506 and nontargeted cells 508 is input into column 502 viaan inlet 510. As the mix of cells flows through column 502, target cells506 bind with beads 504. Nontargeted cells 508 tend not to bind withbeads 504 for lack of a complementary affinity structure. As the mixtureflows through column 502 towards transducer 512, beads 504 are free tomove within the fluidized bed of column 502. As beads 504 approach theacoustic field generated by transducer 512, they are blocked by theacoustic edge effect and/or being trapped in the acoustic field. In anycase, beads 504 are prevented from passing to the output of column 502.As target cells 506 bind to beads 504, target cells 506 are preventedfrom exiting column 502 along with the beads 504 to which they arebound. Nontargeted cells 508 are not influenced as strongly by theacoustic field as are beads 504 and can pass through the acoustic fieldand exit column 502.

This affinity technique employed with fluidized bed system 500 can beimplemented on a single-pass basis. System 500 can be configured withthe choice of beads to select for material that passes through and exitscolumn 502, or to select for material that is bound to the beads andretained in column 502. The passed or retained material can bepositively or negatively selected.

Referring to FIG. 6, an affinity separation process 600 is illustrated.Process 600 includes an external incubation step where affinity beadsand cells are combined together to obtain bead complexes. The mix ofbead complexes and uncombined material in a fluid is fed into a column602. As the fluid mix travels along column 602, the bead complexes aredirected into column 602 by an acoustic field generated by transducer604. The uncombined material exits column 602 by passing through theacoustic field. This separation step retains the bead complexes whileremoving a majority of the uncombined material. Once the bead complexesare loaded into column 602, a flush process can be implemented with theintroduction of a buffer fluid at the base of column 602. The remaininguncombined material moves with the buffer fluid through the acousticfield generated by transducer 604. The bead complexes also move with thebuffer fluid along column 602 but are blocked from exiting by theacoustic field.

Process 600 offers a number of features that are advantageous foraffinity separation of materials. For example, binding of targetmaterial to the beads can take place externally, which also permitsflexible incubation steps. The acoustic separation provides a gentle andhigh throughput separation process that quickly reduces the amount ofuncombined material in mix with bead complexes. For example, theseparation process can be completed in less than one hour. Process 600is also flexibly scalable and can handle processing volumes in the rangeof about 10 mL to about 1 L. In addition, all types of beads may be usedin process 600, providing significant flexibility for unique or customaffinity separation processes.

Referring to FIG. 7, a fluidized bed system 700 is illustrated. A column702 is provided, which can be implemented as any of the columnsillustrated in FIGS. 4-6. In a first wash process, column 702 is loadedwith affinity beads. A wash solution is passed through column 702 whileacoustic transducer 704 is on to generate an acoustic field near a topof column 702. The acoustic field retains the affinity beads in column702 while the wash solution passes through to wash the affinity beads. Acapture process is implemented in which cellular material is introducedinto column 702. Target cellular material binds to the affinity beads toform bead complexes and is blocked from exiting column 702 by theacoustic field generated by the acoustic transducer 704. Nontargetedmaterial can pass through the acoustic field and can exit column 702.After the capture process, a flush process is provided where fluid isintroduced to column 702 to flow the nontargeted material out of column702. The bead complexes are retained in column 702 against the fluidflow by the acoustic field generated by the acoustic transducer 704.

System 700 offers a number of advantageous features for affinityseparation processes, including internal bead binding and low shearforces imposed on the material in column 702. The internal bead bindingwith low shear forces can be important when larger beads are used due topotentially greater binding energy that is associated with larger beads.For example, it may take longer, or a greater amount of energy, fortargeted cellular material to be captured by the larger beads. Lowershear forces can thus help to avoid impeding binding with larger beads.System 700 can employ acoustic transducer 704 to create an acoustic edgeeffect, which can lead to improved throughput. For example, theprocesses of binding and separation can be completed in under 2 hours.System 700 is scalable and can handle processing volumes in the range ofabout 10 mL to about 1 L. the fluidized bed employed in system 700 canbe used with beads or with cells for the purposes of affinity separationand/or separation alone.

Referring to FIG. 8, a cell selection system 800 is illustrated. System800 includes a column 802 that is provided with a stirring mechanism804. Stirring mechanism 804 can be implemented as a stir bar near a baseof column 802. An affinity separation process can be implemented insystem 800 using column 802 as a fluidized bed. Column 802 is loadedwith affinity beads, for example in a range of about 10% to about 30%packing. The affinity beads are washed with the introduction of a fluidinto column 802 while the acoustic field is generated by acoustictransducer 806. The fluid exits column 802 while the affinity beads areblocked from exiting by the acoustic field. A mix of cellular materialis introduced into column 802 while acoustic transducer 806 generates anacoustic field near a top of column 802. All of the cellular material isretained in column 802, along with the affinity beads, with theimplementation of the acoustic field. Excess fluid may pass the acousticfield and exit column 802 while the cells and affinity beads are blockedfrom exiting.

During the wash process and the introduction of the cellular material,transducer 806 may be operated in different modes or with differentcharacteristics to, in one case, block the affinity beads from exitingduring the wash process, and in another case, block both of the affinitybeads and the cellular material from exiting. For example, the frequencyused to drive transducer 806 may be different to retain the affinitybeads than the frequency when both the cells and affinity beads areretained.

Once column 802 is loaded with affinity beads and cellular material,stirring mechanism 804 can be employed to agitate column 802. Theagitation contributes to moving the affinity beads and the cellularmaterial within column 802. As the affinity beads and cellular materialmove within column 802 the affinity binding process for targetedmaterial can be enhanced. This incubation step can be implemented withno fluid flow and with transducer 806 being unenergized.

Once the incubation/binding process is completed, the affinitybead/targeted material complexes can be washed, and nontargeted materialcan be removed from column 802. The targeted material may be separatedfrom the affinity beads with a solution provided to column 802 thatpromotes detachment of the targeted material from the affinity beads.For example, the solution can include enzymes (e.g., trypsin) in abuffer. For example, The targeted material may then be removed fromcolumn 802, while the affinity beads are retained with the acousticfield generated by the acoustic transducer 806.

Referring to FIG. 9, an affinity selection process 900 for positiveselection in a straight column with a single pass is illustrated.Process 900 begins with the loading of column 902 with affinity beadsand washing the beads. Acoustic transducer 904 generates an acousticfield near a top of column 902 during the loading and washing processes.A mix of cellular material is then fed into column 902. Target materialis bound to the affinity beads to form bead complexes. The nontargetedmaterial exits column 902 through the acoustic field. The targetedmaterial is retained with affinity beads in column 902, while thenontargeted material exits column 902. The bead complexes are washedwith the introduction of a buffer into column 902. A detachment bufferis introduced to column 902 to cause the targeted material to detachfrom the affinity beads. With the acoustic field in place, the detachedtargeted material exits column 902 and is collected, while the affinitybeads are retained.

Referring to FIG. 10, an affinity selection process 1000 for negativecell selection in a straight column with a single pass is illustrated.Process 1000 begins with the loading of column 1002 with affinity beadsto a desired void fraction. The loading process can be implemented whileacoustic transducer 1004 is removed from column 1002. With acoustictransducer 1004 connected to a top of column 1002, the affinity beadsare washed with the introduction of a buffer. This washing process alsoserves to expand the bead volume to form a fluidized bed. With acoustictransducer 1004 generating an acoustic field, a mix of cellular materialis fed into column 1002. Target material is bound to the affinity beadsto form bead complexes. The nontargeted material exits column 1002through the acoustic field. The targeted material is retained with theaffinity beads in column 1002, while the nontargeted material exitscolumn 1002 and is collected as the desired product. This negative cellselection removes the targeted material from the mix of cellularmaterial in a single pass. The affinity beads can be multiplexed orconfigured to bind with more than one type of targeted material, whichpermits multiplexed negative selection in a single pass.

Referring to FIG. 11, a graph 1100 illustrates bead retention with anacoustic field versus fluid inflow rate for an acoustic fluidized bedcolumn. As shown in graph 1100, 100% of the beads are retained in thecolumn as the fluid inflow rate increases from 0 to about 10 mL perminute. As the fluid inflow rate increases beyond 10 mL per minute, moreand more beads pass through the acoustic field. The data presented ingraph 1100 is useful to understand the breakthrough fluid inflow ratethat causes beads to pass through the acoustic field. This test used SPSepharose “Fast Flow” beads with an average diameter of 90 um and anaverage density of 1.033 g/cc. The terminal velocity was 52.2 cm/hr. Thecolumn parameters were: volume—40 ml; height—20 cm; and diameter—1.6 cm.The expanded void fraction was 70% with a starting bead concentration of7.86E+05 cells/ml. Operating parameters were: frequency—1 MHz andpower—3 W.

Referring to FIG. 12, a graph 1200 illustrates total viable cellsrecovered in an acoustic affinity system versus column volumes wherecolumn volumes indicates the amount of input to the system normalized bythe volume of the column. As shown in graph 1200, the total viablecells, in millions of cells per milliliter, increases significantlyafter about a half a column volume. This data shows the efficiency ofbinding in the acoustic affinity system. For example, almost no unboundcells are observed during the initial half a column volume of supplyinga cellular material feed to the fluidized bed column.

Referring to FIG. 13, a graph 1300 illustrates a histogram of beadsexiting a fluidized bed column in accordance with particle diameter.Graph 1300 shows that at lower flowrates, small particles escape thecolumn while larger particles are retained. In addition, the averagesize of an escaping particle increases with flow rate.

Referring to FIG. 14, a fluidized bed system 1400 for implementingacoustic affinity cell selection with the recirculation is illustrated.System 1400 includes a column 1402 and an acoustic transducer 1404.Column 1402 includes annular ribs 1406 that can impede the flow of fluidand force fluid flow toward the center of column 1402. Ribs 1406 canhelp prevent undesired effects such as channeling within column 1402.

System 1400 is operated similarly to those discussed above. For example,system 1400 may be used for positive or negative selection and canemploy different modes of operation with the acoustic transducer 1404.System 1400 illustrates the use of recirculation to improve target cellrecovery, by providing more opportunities for target cells to bind withbeads in column 1402. After the beads are loaded into column 1402 andwashed, a pass 1 feed is supplied to column 1402. The outflow of column1402 resulting from the pass 1 feed is collected for use as a pass 2feed. The pass 2 feed is used as the input for a feed supply in afollow-on recirculation pass. Although not shown, the pass 2 feed cangenerate an outflow that can be collected for another follow-onrecirculation pass. Any number of recirculations can be employed. Eachof the example systems and fluidized beds discussed herein can beconfigured to have multiple recirculation passes.

Referring to FIG. 15, a graph 1500 illustrates the purity (P) andpercentage recovery (R) in a fluidized bed system with a number ofrecirculated feed passes. Graph 1500 shows that purity is maintained ata high level, greater than 90% for recirculation passes 1 and 2, andgreater than 80% for recirculation pass 3. The recovery of cellsincreases with each recirculation pass, nearing 100% with the thirdrecirculation.

Several experimental tests for acoustic affinity cell separation wereconducted. The results of the tests are tabulated as examples below.

Example 1

Four fluidized bed platform tests were performed with different cellconcentrations (100 e6/mL and 10 e6/mL) and different capturing antibodycombinations (Anti-TCR a/b only vs Anti-TCR a/b and Anti-CD52) on thefirst day of testing. The initial feed concentration (100 e6/mL and TCRa/b-population was 74-78%).

All samples were incubated with the corresponding capturing antibodycombinations listed in Table 1 in 2% BSA in PBS for 20 minutes on theIKA roller (30 rpm). Cells were washed twice with 2% BSA in PBS andfinally re-suspended in 10 mL 2% BSA in PBS. A sample was removed forflow cytometry and used as the initial population for tests A through D.Next, 4 ml of a 50% solid Promega bead slurry were loaded into thefluidized bed column and washed with 30 ml of a 2% BSA in PBS solutionto remove residual ethanol and particulates. This initial washing stepwas performed at a flow rate and power of 1 ml/min and 0.75 W.

The feed cell population was then separated using the fluidized bed unitpacked with avidin-conjugated methacrylate beads (Promega) whichoperated at the following conditions; flow rate—1 mL/min and power—0.75W. The first fraction, denoted as the outflow, was collected after theentire sample was loaded into the fluidized bed. A second fraction,denoted as the flush, was collected after flushing the fluidized bedwith 30 ml of a 2% BSA in PBS solution at 1 ml/min and 0.75 W. Thisflushing step is implemented to ensure all uncaptured cells arerecovered. Once this process was completed, the remaining contents ofthe column were retrieved and collected as the third fraction, denotedas the holdup. Samples from all three fractions were collected for flowcytometry. For the purposes of conducting a mass balance, the mass andcell count for each fraction was recorded.

TABLE 1 Fluidized Bed (FB) platform test parameters Day 1, Fluidized Bed(FB) test parameters Cell conc. Sample Total cell # Bead TCR a/b CD52Label Antibody Bead [×10{circumflex over ( )}/mL] volume [mL][×10{circumflex over ( )}6] volume [mL] [mL] [mL] Analytics FB_A TCR a/bPromega 100 10 1000 2 1.5 — Counting & Flow FB-B TCR a/b and CD 52Promega 100 10 1000 2 1.5 0.56 Counting & Flow FB-C TCR a/b Promega 1010 100 2 0.15 — Counting & Flow FB-D TCR a/b and CD52 Promega 10 10 1002 0.15 0.06 Counting & Flow

The purity increased by approximately 15% for all samples afterseparation by the fluidized bed unit, where the initial cell populationconsisted of 76% TCR knockout cells. In tests conducted at a higher cellconcentration (100E6 cells/mL), samples A and B yielded a purity of 13%and 10% in the outflow and 11% and 8% in the flush respectively showinga slight decrease in purity in the second fraction. The purity in theholdup fraction was 73.2% and 68.1% for samples A and B indicating that100% purity was not achieved. Tests conducted at lower cellconcentrations (10E6 cells/mL) yielded a higher purity of 90.5% and92.4% in the outflow fraction and even higher purity in the flushfraction of 94.8% and 93.2% in samples C and D respectively. This resultshowed that lower cell concentrations were better with currentconditions used with the fluidized bed unit. Overall employing thecombination of anti-TCR and anti-CD52 as capturing antibodies did notyield significantly different purity compared to using anti-TCR as thesole capturing antibody.

TABLE 2 TCR a/b-purity and recovery from Fluidized Bed test Day 1,Fluidized Bed (FB) test restults TCR a/b-purity [%] Recovery LabelControl Outflow Flush Hold-up [%] FB_A 74.50% 84.10% 83.10% 73.00% 6.70%FB_B 75.40% 83.10% 81.90% 67.50% 4.80% FB_C 78.10% 90.50% 94.80% 78.40%33.50% FB_D 76.30% 92.30% 93.20% 81.30% 32.90%

The total TCR-recovery for each test is equal to the sum of TCR-cells inthe flow-through and flush fractions divided by the starting TCR-cellcount (See Eq. 6 in Appendix). There are two mechanisms by whichTCR-cells could be retained in the fluidized bed system: acousticretention and inefficient flushing. Acoustic retention occurs when afree cell experiences a greater force from the acoustic field comparedto the drag force exerted by the fluid flow. This happens at high powerto flow rate ratios and can be prevented by optimizing operatingconditions. Cells also tend to disperse into the volume of the system,making a flush step necessary to improve recovery. The flushing stepshould have a uniform velocity distribution, otherwise a large volume ofbuffer is needed to recover TCR-cells as the incoming wash buffer mixeswith the fluidized bed. This type of cell retention can be reduced byincreasing flush velocity and volume and improving the fluidized bedinlet design.

For each fluidized bed test the total TCR-cell recovery can be seen inTable 2.

The lowest recoveries were seen while testing high cell densities (100e6/ml). Tests A and B had TCR-recoveries of 7% and 5% respectively. A“clogging” effect in the column was observed during these tests wherebeads and cells agglomerated together in very large clumps. Rather thanacting as a fluid these solid clumps caused channeling in the column andprevented cells from escaping. It is also possible that non-specificbinding occurred as the column fouled.

Tests C and D had similar recoveries, 34% and 33%. The two tests with 10e6/ml behaved as expected but still had relatively low TCR-cellrecoveries. This is due to the low fluid velocity and inefficient flushstep described previously and can be improved by optimizing operatingconditions and improving the fluidized bed inlet design. Changing theantibody had a minimal effect on cell recovery.

Example 2

Four Acoustic Separator unit tests were performed with differentaffinity bead types (Promega, Dynabead, PolyStyrene 6 um and 14 um). OnDay 2, fixed antibody combination (Anti-TCR and Anti-CD52) and antibodyvolume of 0.15 mL and 0.052 mL, respectively) were used. The initial TCRa/b-population was 77%.

All samples were incubated with anti-TCR and anti-CD52 in 2% BSA in PBSfor 20 minutes on the IKA roller (30 rpm). Cells were washed twice with2% BSA in PBS. A sample was removed for flow cytometry and used as theinitial population for tests L through Q. Samples were incubated withthe corresponding bead candidate listed in Table 3. for 30 minutes onthe IKA roller (30 rpm) in 10 mL of 2% BSA in PBS and then separatedusing the Acoustic Separator unit operated at the following conditions;flow rate—1 mL/min and power—0.75 W. The first fraction, denoted as theoutflow was collected after the entire sample passed through theacoustic field. A second fraction denoted as the flush was collectedafter flushing the fluidized bed with 30 ml of a 2% BSA in PBS solution.Once this process was completed, the remaining contents of the columnwere retrieved and collected as the third fraction, denoted as theholdup. Samples from all three fractions were collected for flowcytometry. For the purposes of conducting a mass balance, the mass andcell count for each fraction was recorded.

TABLE 3 Acoustic Separator (AC) platform test parameters Day 2, AcousticSeparator (AC) parameters Cell conc. Sample volume Total cell # Beadvolume TCR a/b CD52 Label Antibody Bead [×10{circumflex over ( )}/mL][mL] [×10{circumflex over ( )}6] [mL] [mL] [mL] Analytics AC_A TCR a/band CD52 Promega 100 10 100 2 0.15 0.056 Counting & Flow AC_B TCR a/band CD52 Dynabead 100 10 100 0.015 0.15 0.056 Counting & Flow AC_C TCRa/b and CD52 PS (6um) 10 10 100 0.015 0.15 0.056 Counting & Flow AC_DTCR a/b and CD52 PS (14um) 10 10 100 0.015 0.15 0.056 Counting & Flow

The purity increased by approximately 13% for all samples afterseparation by the Acoustic Separator unit, where the initial cellpopulation consisted of 77% TCR knockout cells. The sample incubatedwith Dyna beads resulted in the highest purity of 89.4% in the outflowfraction while the sample incubated with Polystyrene (10-14 μm) beadsresulted in the lowest purity of 84.3%. This trend was also observed inthe flush fraction where samples incubated with Dyna beads yielded 91.1%purity and Polystyrene beads yielded 84.5% purity. The purity in allsamples increased slightly from the outflow (84.3%-89.8%) to the flushfraction (84.6%-91.1%).

TABLE 4 TCR a/b-purity and recovery from Fluidized Bed test Day 2,Acoustic Separator (AC) test restults TCR a/b-purity [%] Recovery LabelControl Outflow Flush Hold-up [%] AC_A 76.20% 85.70% 86.20% 79.20%79.90% AC_B 77.60% 89.80% 91.10% 88.90% 17.00% AC_C 77.70% 88.30% 89.40%84.70% 26.20% AC_D 77.70% 84.30% 84.60% 79.70% 27.10%

The total TCR-cell recoveries for each acoustic separator system testcan be seen in Table 4. In this figure it appears that the recovery isaffected by the bead type, with 50 um Promega beads having an 80%TCR-cell recovery and 4.5 um Dyna-beads having just 17% recovery. Bothpolystyrene particles had similar recoveries, 26% and 27% for 6 um and14 um beads respectively. Since every test was performed with the sameoperating conditions, similar recoveries were expected so thisrelationship should be confirmed in future work. Like in the fluidizedbed, TCR-recovery in the Acoustic Separator system can be increased byincreasing flow velocity and by improving the inlet and collectordesigns.

Example 3

Two Fluidized Bed (FB) processes and two Acoustic Separator (AS)processes were performed. Different pump systems (Syringe pump andperistaltic pump) were tested on the Fluidized Bed unit and two new beadcandidates were tested on the Acoustic Separator unit on the first dayof testing. The initial feed concentration was 10⁷ cells/mL and TCRa/b-population was about 80%.

Sample preparation and acoustic unit operating procedures were the sameas previous examples. Briefly, feed samples for the Fluidized Bed wereincubated with biotinylated anti-TCR a/b antibody (Table 5) in 2% BSA inPBS for 20 minutes on the IKA roller (30 rpm). Cells were washed twicewith 2% BSA in PBS and finally re-suspended in 10 mL 2% BSA in PBS. Forthe feed samples for the Acoustic Separator unit, bead incubation wasfollowed by antibody-cell incubation. 1×10⁶ cells from each feed samplewere collected separately for flow cytometry and used as the initialpopulation for each test.

The fluidized bed column was loaded with 2 ml Promega bead slurry(avidin-conjugated methacrylate beads) and then washed with 30 ml of a2% BSA in PBS solution to remove residual ethanol and particulates. Thisinitial washing step was performed at 3 mL/min and 2.25 W. Two differentpumps (Syringe pump—FB_A and Peristaltic pump—FB_B) were evaluated onday 1. The feed cell population was then separated using the fluidizedbed unit packed with Promega beads which operated at 3 mL/min and 4 mLcolumn volume. For the Acoustic Separator unit operation, bead labeledfeed were separated at the following conditions; flow rate—1 mL/min andpower—0.75 W.

For the performance evaluation, processed samples from both units werecollected and analyzed from three different fractions—outflow, flush andholdup (Table 6). The first fraction of the processed sample, denoted asthe outflow, was collected after the entire sample was loaded into thefluidized bed. A second fraction, denoted as the flush, was collectedafter flushing the fluidized bed with 30 ml of 2% BSA in PBS solution.This flushing step is necessary to ensure all uncaptured cells arerecovered. Once this process was completed the remaining contents of thecolumn were retrieved and collected as the third fraction, denoted asthe holdup. Samples from all three fractions were collected for flowcytometry. The mass and cell concentration count for each fraction wasrecorded for the cell recovery evaluation.

TABLE 5 Fluidized Bed (FB) and Acoustic Separator (AS) unit testparameters (bead volume = 1 cc, slurry volume = 4 ml. Day 1, FluidizedBed (FB) and Acoustic Separator (AS) test, parameters Cell conc. Samplevolume Bead volume TCR a/b Power Flow Rate Label Antibody Bead[×10{circumflex over ( )}/mL] [mL] [mL] [mL] [W] [mL/min] Comments FB_AAnti-TCR Promega 100 10 2 0.15 2.25 3 Syrinage pump FB_B Anti-TCRPromega 100 10 2 0.15 2.25 3 Peristaltic pump AS_C Anti-TCR PLGA 10 100.15 0.15 0.75 1 Syrinage pump AS_D Anti-TCR WAX 10 10 0.15 0.15 0.75 1Syrinage pump

The purity increased by approximately 11˜12% for all samples afterseparation by the Fluidized Bed unit and almost no change afterseparation by the Acoustic Separator unit, where the initial cellpopulation consisted of 80% TCR knockout cells (Table 6). For theFluidized Bed tests, both peristaltic pump (FB_A) and syringe pump(FB_B) resulted in similar level of purity (90˜92%) in the flow throughand flush fraction. In addition to fluidized bed testing, two differentmicron sized bio-degradable particle candidates (AS_A—PLGA and AS_B—Wax)were tested in Acoustic Separator unit.

Table 6 also shows recovery results. Fluidized Bed with peristaltic pump(FB_A) and syringe pump (FB_B) showed 78% and 61% of TCR—recovery,respectively. Based on the results, FDS decided to use peristaltic pumpfor upcoming platform validation. Peristaltic pump enables flexibilityof further process optimization and closed system development. AcousticSeparator for PLGA and Wax resulted low recovery (50% and 38%,respectively).

TABLE 6 Fluidized Bed(FB) and Acoustic Separator (AS) unit testparameters Day 1, Fluidized Bed (FB) and Acoustic Seperator (AS) test,results Processed TCR a/b-[%] Recovery Label Control Flow through FlushHold-up [%] FB_A 80.20% 92.24% 91.44% 86.90% 78.04% FB_B 79.10% 90.43%91.26% 73.20% 61.37% AS_A 80.00% 80.40% 80.10% 79.50% 49.54% AS_B 80.60%81.00% 83.70% 79.70% 38.49%

Example 4

Four fluidized bed unit tests were performed with different operationprocedures. The residence time of feed cells in the column was increasedby re-circulation of the processed sample or by holding samples in thecolumn for a longer time period. The initial feed concentration was 10⁷cells/mL and TCR a/b-population was about 80%.

The same procedure was performed for feed and initial bead loading ofthe Fluidized Bed unit as in day 1. Table 7 shows four differentoperation procedures, no recirculation (FB_E, no recirc.), onerecirculation (FB_F, 1 recirc.), 4 recirculations (FB_G, 4 recirc.) andstop and flow (FB_H, Stop and Flow). Specifically, in the stop and flowcondition, 2.5 mL of feed samples were loaded with (3 mL/min) and flowstopped for 3 min 20 sec. This procedure was repeated until all the feedvolume was loaded into the column. All the feed cells were held in thecolumn by higher power condition (4.5 W) for a total of 13 min 20 sec.Once the recirculation steps and stop and flow steps were finished, thecolumn was flushed with 30 mL of 2% BSA solution. The processed sampleswere collected and analyzed as in day 1.

TABLE 7 Fluidized Bed (FB) platform and Acoustic Separator (AS) unittest parameters (bead volume = 1 ml) Day 2, Fluidized Bed (FB) test,parameters Cell conc. Sample volume Bead volume TCR a/b Power Flow RateLabel Antibody Bead [×10{circumflex over ( )}/mL] [mL] [mL] [mL] [mL][mL] Comments FB_E TCR Promega 10 10 2 0.15 2.25 3 No recirc. FB_F TCRPromega 10 10 2 0.15 2.25 3 1 recirc. FB_G TCR Promega 10 10 2 0.15 2.253 4 recirc. FB_H TCR Promega 10 10 2 0 15 2.25 3 Stop and Flow

The purity increased by approximately 9-18% for all samples afterseparation, where the initial cell population consisted of 80% TCRknockout cells (Table 8). One recirculation (FB_F) resulted 95.6% and97.7% of purity in Flow through and Flush portion, respectively.Notably, 4 recirculations (FB_G) showed low purity and we observed sometemperature increase due to the 4 times of recirculation. Thetemperature rising also happened in stop and flow condition (FB_H).

TABLE 8 TCR a/b-purity and recovery from Fluidized Bed and AcousticSeparator test Day2, Fluidized Bed (FB) test, results Processed TCRa/b-[%] Recovery Label Control Flow through Flush Hold-up [%] FB_E89.50% 93.16% 96.37% 73.50% 51.86% FB_F 79.80% 95.64% 97.69% 59.20%82.64% FB_G 80.50% 89.50% 84.80% 56.10% 98.89% FB_H 80.10% 95.11% 91.51%73.60% 85.75%

For the TCR a/b-recovery, Recirculation and Stop and Flow conditionshowed good results. Adding more recirculation steps showed betterrecovery (one recirc.—82.64% and 4 recirc. 98.89%) and stop and flowcondition also resulted high recovery (85.75%).

In accordance with the present disclosure, an acoustic affinity systemis discussed that provides a number of advantageous features. Forexample, the systems and methods discussed herein can provide increasedrecovery and purity for target cellular material. The systems andmethods are scalable, capable of handling a relatively wide range ofmaterial volumes. Positive and negative selection can be implemented inaccordance with the present disclosure. Positive selection can includeimplementations with apheresis products. Negative and positive selectioncan be implemented on a multiplexed basis, where multiple types ofcellular material can be selected in one pass. The systems and processesdiscussed herein can be fully automated and can be figured to be usedwith consumable components. The acoustic affinity cell selection systemcan be integrated with a cellular concentrate-wash device and/or systemfor downstream applications.

The methods, systems, and devices discussed above are examples. Variousconfigurations may omit, substitute, or add various procedures orcomponents as appropriate. For instance, in alternative configurations,the methods may be performed in an order different from that described,and that various steps may be added, omitted, or combined. Also,features described with respect to certain configurations may becombined in various other configurations. Different aspects and elementsof the configurations may be combined in a similar manner. Also,technology evolves and, thus, many of the elements are examples and donot limit the scope of the disclosure or claims.

Specific details are given in the description to provide a thoroughunderstanding of example configurations (including implementations).However, configurations may be practiced without these specific details.For example, well-known processes, structures, and techniques have beenshown without unnecessary detail to avoid obscuring the configurations.This description provides example configurations only, and does notlimit the scope, applicability, or configurations of the claims. Rather,the preceding description of the configurations provides a descriptionfor implementing described techniques. Various changes may be made inthe function and arrangement of elements without departing from thespirit or scope of the disclosure.

Also, configurations may be described as a process that is depicted as aflow diagram or block diagram. Although each may describe the operationsas a sequential process, many of the operations can be performed inparallel or concurrently. In addition, the order of the operations maybe rearranged. A process may have additional stages or functions notincluded in the figure.

Having described several example configurations, various modifications,alternative constructions, and equivalents may be used without departingfrom the spirit of the disclosure. For example, the above elements maybe components of a larger system, wherein other structures or processesmay take precedence over or otherwise modify the application of theinvention. Also, a number of operations may be undertaken before,during, or after the above elements are considered. Accordingly, theabove description does not bound the scope of the claims.

A statement that a value exceeds (or is more than) a first thresholdvalue is equivalent to a statement that the value meets or exceeds asecond threshold value that is slightly greater than the first thresholdvalue, e.g., the second threshold value being one value higher than thefirst threshold value in the resolution of a relevant system. Astatement that a value is less than (or is within) a first thresholdvalue is equivalent to a statement that the value is less than or equalto a second threshold value that is slightly lower than the firstthreshold value, e.g., the second threshold value being one value lowerthan the first threshold value in the resolution of the relevant system.

What is claimed is:
 1. A separation system, comprising: an expanded bedcolumn with an opening on each end to permit a fluid flow therethrough;and an acoustic transducer on one end of the column for generating anacoustic standing wave in the column; and the acoustic transducer beingconfigured to generate the acoustic standing wave to block or permitpassage of material entrained in the fluid flow.
 2. The system of claim1, further comprising an agitator coupled to the column for agitatingthe fluid.
 3. The system of claim 1, wherein the acoustic transducer isconfigured to operate in a plurality of modes.
 4. The system of claim 3,wherein the plurality of modes comprise a clustering mode and an edgeeffect mode.
 5. The system of claim 1, wherein the acoustic transduceris configured to generate a multi-dimensional acoustic standing wave inthe column.
 6. The system of claim 1, further comprising mobile supportstructures for cellular material in the column that are responsive tothe acoustic standing wave.
 7. The system of claim 6, wherein thesupport structures comprise affinity beads.
 8. The system of claim 7,wherein the affinity beads comprise column packing in a range of fromabout 10% to about 30%.
 9. The system of claim 7, wherein the affinitybeads are configured with one or more of anti-TCR or anti-CD52 capturingantibodies.
 10. The system of claim 7, wherein the affinity beads areconfigured as avidin-conjugated methacrylate beads.
 11. A method forseparating materials, comprising: providing support structures in anexpanded bed column for binding with a first material; flowing a fluidmixture that includes the first material into the column; and generatingan acoustic standing wave with an acoustic transducer near an end of thecolumn to block the support structures from leaving the column with thefluid flow.
 12. The method of claim 11, further comprising agitating thefluid mixture in the column.
 13. The method of claim 11, furthercomprising generating the acoustic standing wave in a plurality ofmodes.
 14. The method of claim 13, wherein the plurality of modescomprise a clustering mode and an edge effect mode.
 15. The method ofclaim 11, further comprising generating a multi-dimensional acousticstanding wave in the column.
 16. The method of claim 11, wherein thesupport structures comprise affinity beads.
 17. The method of claim 16,further comprising packing the column in a range of from about 10% toabout 30% with the affinity beads.
 18. The method of claim 16, whereinthe affinity beads are configured with one or more of anti-TCR oranti-CD52 capturing antibodies.
 19. The method of claim 16, wherein theaffinity beads are configured as avidin-conjugated methacrylate beads.20. An acoustic affinity separation method, comprising: providingaffinity beads in a fluid to a column; generating an acoustic standingwave with an acoustic transducer near an end of the column; flowing acellular material fluid mixture in the column and through the acousticstanding wave; configuring the acoustic standing wave to prevent theaffinity beads from passing through the acoustic standing wave.